PMC:5026484 / 15875-18170
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/5026484","sourcedb":"PMC","sourceid":"5026484","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5026484","text":"Some bMERB domains can bind two Rab proteins simultaneously\nBesides the specificity of bMERB domains towards the Rab8 family, another interesting observation was made in the ITC experiments comparing the stoichiometry of binding of Rab8 and the different RBDs: Whereas Rab8 bound in a 1:1 stoichiometry to Mical-cL and EHBP1, a 2:1 stoichiometry binding was observed for Mical-1, Mical-3 and EHBP1L1 (Table 1 and Figure 2b). For Rab8 and Mical-1/Mical-3, the ITC experiments show one high-affinity enthalpy-driven and one lower affinity entropy-driven binding site, compared to two binding sites with similar affinity for Rab8 and EHBP1L1.\nIn order to confirm the observed differences, we repeated the aSEC experiments with varying ratios of Rab8 and the different RBDs of Micals and EHBPs (1.2:1, 1.6:1 and 2.2:1; Figure 2c). These experiments clearly confirmed the aforementioned differences in the stoichiometry of binding with a 2:1 stoichiometry being observed for the Rab8:EHBP1L1 and Rab8:Mical-1 complexes, but not for others tested. The low affinity second binding site of Mical-3 (KD = 4.4 µM as determined by ITC, Table 1) could also not be detected in these experiments, suggesting dynamic complex formation with a large koff. These data show that Mical-1 and EHBP1L1 contain two binding sites that bind Rab8 with high affinity, whereas Mical-3 (and possibly Mical-cL and EHBP1) contain one high affinity and a second lower affinity binding site for Rab8.\nThe presence of two distinct Rab binding sites on certain bMERB domains was a striking observation pointing towards a possible function of these effector proteins in sorting cargo and/or linking different endosomal trafficking pathways regulated by different Rab proteins. In accordance with this idea, recent studies on Mical-L2 dependent GLUT4 translocation showed that trafficking was dependent on a concerted action of Rab8 and Rab13 (Sun et al., 2010, 2016). We consequently also tested whether the effector proteins might be able to simultaneously bind two different Rab proteins in a 1:1:1 (RabX:effector:RabY) complex using both Rab8 and Rab13 and the corresponding aSEC experiments clearly confirmed the formation of a ternary complex of Rab8:Mical-1:Rab13 as well as Rab8:EHBP1L1:Rab13 (Figure 2—figure supplement 3).","divisions":[{"label":"Title","span":{"begin":0,"end":59}}],"tracks":[{"project":"2_test","denotations":[{"id":"27552051-21041651-26907293","span":{"begin":1919,"end":1923},"obj":"21041651"},{"id":"27552051-26538022-26907294","span":{"begin":1925,"end":1929},"obj":"26538022"}],"attributes":[{"subj":"27552051-21041651-26907293","pred":"source","obj":"2_test"},{"subj":"27552051-26538022-26907294","pred":"source","obj":"2_test"}]},{"project":"MyTest","denotations":[{"id":"27552051-21041651-26907293","span":{"begin":1919,"end":1923},"obj":"21041651"},{"id":"27552051-26538022-26907294","span":{"begin":1925,"end":1929},"obj":"26538022"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"attributes":[{"subj":"27552051-21041651-26907293","pred":"source","obj":"MyTest"},{"subj":"27552051-26538022-26907294","pred":"source","obj":"MyTest"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93c2","default":true},{"id":"MyTest","color":"#93dcec"}]}]}}